1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect haptoglobin.
2. Description of the Prior Art
Haptoglobin is a macromolecular glycoprotein which is the major acute phase reactant in cattle and other ruminants [Bremner, 1964, Aust. J. Exp. Biol. Med. Sci., 42:643-656; Goodger, 1970, Clin. Chim. Acta., 29:429-435; Blakeslee and Stone, 1971, Vox Sang., 21:175-182; Spooner and Miller, 1971, Vet. Rec., 12:2-4; Putnam, 1975, Haptoglobin, In: The Plasma Proteins, Structure, Function, and Genetic Control, 11, Putnam (ed.), Academic Press, New York, pp. 1-50; Blackshaw, 1979, NZ Vet. J., 27:103-105; Eckersall and Conner, 1990, Comp. Biochem. Physiol., 96B:309-314; and Morimatsu et al., 1991, J. Biol. Chem., 266:11833-11837].
Biochemical studies have shown that bovine haptoglobin is composed of two chains, an alpha-chain and a beta-chain. The reported molecular weights of the chains has varied among investigators, with the alpha-chain having been described as 16 and 23 KDa, and the beta-chain having been described as 40 and 35 KDa [Morimatsu et al., ibid, Eckersall and Conner, ibid]. Two alpha-chains and two beta-chains are linked by a disulfide bond, forming the basic tetrameric subunit of the compound. In biological material such as blood, haptoglobin binds to hemoglobin, forming a stable complex therewith [Putnam, ibid; Morimatsu et al., ibid]. Eckersall and Conner (ibid) have also reported that haptoglobin is associated with albumin in bovine plasma. The basic tetrameric subunit and albumin associate into polymers of varying molecular weight. Eckersall and Conner have reported molecular weights of 670 KDa and Morimatsu et al. have reported variable molecular weights up to 2000 KDa.
As an acute phase reactant, haptoglobin serum levels become greatly elevated immediately following injury or infection. Clinically, less than 5% of healthy cattle have measurable levels [10 mg % hemoglobin binding capacity (HbBC)] of haptoglobin in their serum (Bremner; Goodger; Blakeslee and Stone; Spooner and Miller; Blackshaw; and Eckersall and Conner, all ibid). However, a rapid increase in the serum haptoglobin level occurs following infection, inflammation or trauma, with serum levels exceeding 100 mg % HbBC of haptoglobin within 3 to 4 days following trauma (Spooner and Miller, ibid). Haptoglobin therefore represents a nonspecific, highly sensitive indicator of disease or tissue damage in cattle [Liberg, 1977, Acta Vet. Scand., 18:335-348; Dinarello, 1984, Induction of Acute Phase Reactants by Interleukin-1, IN: Advances in Inflammation Research, 8, Weissman (ed.), Raven Press, New York, pp. 203-225; Conner et al., 1986, Res. Vet. Sci., 41:126-128; and Eckersall and Conner, 1988, Vet. Res. Commun., 12:169-178].
Considerable research has dealt with illuminating the conditions that stimulate the production of haptoglobin and the in vivo time course of haptoglobin production [Blakeslee and Stone; Spooner and Miller; Liberg; Blackshaw; Dinarello; Conner et al. 1986; and Eckersall and Conner 1988, all ibid; and Conner et al., 1986 Prot. Biol. Fluids, 34:509-512; Conner et al., 1988, Res. Vet. Sci., 44:82-88; Conner et al., 1989, Res. Vet. Sci., 47:203-207; Erskine and Ridell, 1990, The Acute Phase Response During Experimental Escherichia coli Mastitis, In: The International Symposium on Bovine Mastitis, Sep. 13-16, 1990, Indianapolis, Ind., pp. 60-63; and Makimura and Usui, 1990, Jpn. J. Vet. Sci., 52(6):1245-1250]. Additional studies have focused on the purification and biochemical characterization of haptoglobin [Owen et al., 1960, J. Clin. Pathol., 13:163-164; Bremner, ibid; Goodger 1970, ibid; Goodger, 1972, Aust. J. Exp. Biol. Med. Sci., 50:11-20; Javid and Liang, 1973, J. Lab. Clin. Med., 82:991-1002; Spooner, 1973, Res. Vet. Sci., 4:90-96; Makimura and Suzuki, 1982, Jpn. J. Vet. Sci., 44:15-21; Osada, 1985, Acta Biochim. Pol., 32:225-233; Eckersall and Conner 1990, ibid; Morimatsu et al., ibid; Skinner et al., 1991, Vet. Rec., 128:147-149; and Yoshino et al., 1992, Am. J. Vet. Res., 53(6):951-956].
There exists a need for an indicator of preclinical illness in cattle to reduce the number of carcasses condemned after slaughter. However, the use of serum haptoglobin levels as this diagnostic tool has been hampered by the lack of a simple, rapid, and sensitive assay for determining haptoglobin concentrations. The current method used to quantify haptoglobin is a colorimetric procedure based upon the difference in peroxidase activity between free hemoglobin and the hemoglobin-haptoglobin complex (Owen et al.; and Makimura and Suzuki, both ibid). Unfortunately, this is a cumbersome assay, and the accuracy of the method is dependent upon the quality of the sample, particularly the collection of plasma that is free of hemoglobin contamination.